U.S. patent number 7,820,374 [Application Number 10/301,498] was granted by the patent office on 2010-10-26 for detection methods based on hr23 protein binding molecules.
This patent grant is currently assigned to Erasmus Universiteit Rotterdam. Invention is credited to Steven Bergink, Jan H. J. Hoeijmakers, Mei Yin Ng, Gijsbertus Theodoras Johannes van der Horst, Wim Vermeulen.
United States Patent |
7,820,374 |
Hoeijmakers , et
al. |
October 26, 2010 |
**Please see images for:
( Certificate of Correction ) ** |
Detection methods based on HR23 protein binding molecules
Abstract
A method is provided for determining whether an agent is capable
of inducing a DNA lesion in a eukaryotic cell, including exposing
the eukaryotic cell to the agent and determining whether an HR23
protein-binding molecule accumulates in the cell, where the HR23
protein-binding molecule is preferably xeroderma pigmentosum group
C (XPC), 3-methyladenine DNA glycosylase (MAG), CREB, p53, or a
functional part, derivative, and/or analogue thereof. Preferably
the cell overexpresses HR23A and/or HR23B protein. A rapid and
sensitive test is provided with significant advantages over the
Ames test. A method is provided for determining whether an agent is
capable of inhibiting a cellular process, the process resulting in
accumulation of HR23 protein-binding molecule within a cell. A
method for determining whether a cell has an impaired DNA repair
system is provided. An impaired DNA repair system is indicative for
diseases such as xeroderma pigmentosum, cockayne syndrome, and/or
trichothiodystrophy.
Inventors: |
Hoeijmakers; Jan H. J.
(Zevenhuizen, NL), Bergink; Steven (Rotterdam,
NL), van der Horst; Gijsbertus Theodoras Johannes
(Rhoon, NL), Vermeulen; Wim (Zwijndrecht,
NL), Ng; Mei Yin (Philadelphia, PA) |
Assignee: |
Erasmus Universiteit Rotterdam
(Rotterdan, NL)
|
Family
ID: |
26972406 |
Appl.
No.: |
10/301,498 |
Filed: |
November 20, 2002 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20030124605 A1 |
Jul 3, 2003 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60331773 |
Nov 21, 2001 |
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Current U.S.
Class: |
435/6.16; 435/8;
435/29; 435/7.1 |
Current CPC
Class: |
G01N
33/6872 (20130101); C12Q 1/6827 (20130101); G01N
33/5014 (20130101); G01N 33/5091 (20130101); C12N
15/8509 (20130101); G01N 33/6896 (20130101); G01N
33/502 (20130101); G01N 33/5008 (20130101); A01K
67/0276 (20130101); C12Q 1/6827 (20130101); C12Q
2527/127 (20130101); C12Q 2521/514 (20130101); A01K
2227/105 (20130101); A01K 2267/03 (20130101); C12Q
1/6883 (20130101); A01K 2217/075 (20130101) |
Current International
Class: |
C12Q
1/68 (20060101); G01N 33/53 (20060101); C12Q
1/66 (20060101) |
Other References
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factors in vivo. Molecular Cell. vol. 8, pp. 213-224, Jul. 2001.
cited by examiner .
Fitch et al. p53 responsive nucleotide excision repair gene
products p48 and XPC, but not p53, localize to sites of
UV-irradiation-induced DNA damage, in vivo. Carcinogenesis, vol.
24, pp. 843-850, May 2003. cited by examiner .
Kroese et al. Genetic tests and their evaluation: can we answer the
key questions? Genetics in Medicine, vol. 6, pp. 475-480, 2004.
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examiner .
"irradiate." Merriam-Webster Online Dictionary. 2008.
Merriam-Webster Online. May 22, 2008
<http://www.merriam-webster.com/dictionary/irradiate>. cited
by examiner .
Hoogstraten et al. Versatile DNA damage detection by the global
genome nucleotide excision repair protein XPC. Journal of Cell
Science, vol. 121, pp. 2850-2859, 2008. cited by examiner .
Uchida et al. The carboxy-terminal domain of the XPC protein plays
a crucial role in nucleotide excision repair through interactions
with transcription factor IIH. DNA Repair, vol. 1, No. 6, pp.
449-461, Jun. 2002. cited by examiner .
Masutani et al. Identification and characterization of XPC-binding
domain of hHR23B. Molecular and Cellular Biology, vol. 17, No. 12,
pp. 6915-6923, Dec. 1997. cited by examiner .
Yamaizumi et al., U.v.-induced nuclear accumulation of p53 is
evoked through DNA damage of actively transcribed genes independent
of the cell cycle, Oncogene, 1994, pp. 2775-2784, vol. 9, No. 10.
cited by other .
Batty et al., Stable Binding of Human XPC Complex to Irradiated DNA
Confers Strong Discrimination for Damages Sites, Journal of
Molecular Biology, 2000, pp. 275-290, vol. 300, No. 2. cited by
other .
Hey et al., the XPC-HR23B Complex Displays High Affinity and
Specificity for Damaged DNA in a True-Equilibrium Fluorescence
Assay, Biochemistry, May 28, 2002, pp. 6583-6587, vol. 41, No. 21.
cited by other .
Sugasawa et al., Xeroderma Pigmentosum Group C Protein Complex Is
the Initiator of Global Genome Nucleotide Excision Repair,
Molecular Cell, Aug. 1998, pp. 223-232, vol. 2, No. 2. cited by
other .
Yokoi et al., The Xeroderma Pigmentosum Group C Protein Complex
XPC-HR23B Plays an Important Role in the Recruitment of
Transcription Factor IIH to Damaged DNA, Mar. 2000, pp. 9870-9875,
vol. 275, No. 13. cited by other .
Ng et al., Developmental Defects and Male Sterility in Mice Lacking
the Ubiquitin-Like DNA Repair Gene mHR23B, Molecular and Cellular
Biology, Feb. 2002, pp. 1233-1245, vol. 22, No. 4. cited by other
.
Santagati et al., Different dynamics in nuclear entry of subunits
of the repair/transcription factor TFIIH, Nucleic Acids Research,
Apr. 1, 2001, pp. 1574-1581, vol. 29, No. 7. cited by other .
PCT International Search Report, PCT/NL03/00812, Aug. 17, 2004.
cited by other.
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Primary Examiner: Dunston; Jennifer
Attorney, Agent or Firm: TraskBritt
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority under 35 U.S.C. .sctn.119(e) to
U.S. Provisional Patent Application 60/331,773, filed on Nov. 21,
2001, the contents of the entirety of which are incorporated by
this reference.
Claims
What is claimed is:
1. A method for detecting a DNA lesion that is a substrate for
global genome nucleotide excision repair in a mouse embryonic
fibroblast that is deficient in endogenous HR23A and HR23B and that
comprises an exogenous nucleic acid expressing HR23 protein or a
functional part thereof, the method comprising: determining if
there is an overall intranuclear accumulation of full-length
xeroderma pigmentosum group C (XPC) protein within the mouse
embryonic fibroblast; and correlating said overall intranuclear
accumulation of full-length xeroderma pigmentosum group C protein
with a DNA lesion that is a substrate for global genome nucleotide
excision repair in the mouse embryonic fibroblast.
2. The method according to claim 1, wherein said mouse embryonic
fibroblast is overexpressing HR23A and/or HR23B protein, or a
functional part thereof.
3. The method according to claim 1, wherein said mouse embryonic
fibroblast is part of a cell line.
4. The method according to claim 1, wherein said mouse embryonic
fibroblast is provided with a nucleic acid encoding human HR23A
protein or a functional part thereof.
5. The method according to claim 1, wherein said mouse embryonic
fibroblast is provided with a nucleic acid encoding murine HR23A
protein or a functional part thereof.
6. The method according to claim 1, wherein said mouse embryonic
fibroblast has been provided with a nucleic acid encoding human
HR23B protein or a functional part thereof.
7. The method according to claim 1, wherein said mouse embryonic
fibroblast is provided with a nucleic acid encoding murine HR23B
protein or a functional part thereof.
8. The method according to claim 1, wherein said xeroderma
pigmentosum group C protein comprises a human xeroderma pigmentosum
group C protein.
9. The method according to claim 1, wherein the XPC protein
comprises a label.
10. A method for determining whether an agent is capable of
inducing a DNA lesion that is a substrate for global genome
nucleotide excision repair or capable of at least in part
inhibiting a cellular process, the cellular process resulting in
accumulation of xeroderma pigmentosum group C protein in a mouse
embryonic fibroblast that is deficient in endogenous HR23A and
HR23B and that comprises an exogenous nucleic acid expressing an
HR23 protein or a functional part thereof, the method comprising:
exposing at least one mouse embryonic fibroblast that is deficient
in endogenous HR23A and HR23B and that comprises an exogenous
nucleic acid expressing an HR23 protein or a functional part
thereof to the agent; determining if there is an overall
intranuclear accumulation of full-length xeroderma pigmentosum
group C (XPC) protein within the mouse embryonic fibroblast; and
correlating the overall intranuclear accumulation of full-length
xeroderma pigmentosum group C protein with said agent's ability to
induce a DNA lesion that is a substrate for global genome
nucleotide excision repair or to at least in part inhibit a
cellular process in the mouse embryonic fibroblast; wherein the
cellular process is selected from the group consisting of
proteasomal proteolysis, nucleo-cytoplasm shuttling, and any
combination thereof.
11. The method according to claim 10, wherein the XPC protein
comprises a label.
12. The method according to any one of claims 9 or 11, wherein said
label comprises green fluorescent protein or luciferase.
Description
TECHNICAL FIELD
The invention relates generally to biotechnology, more particularly
to the field of molecular biology.
BACKGROUND
The integrity of a cellular organism is continuously challenged
during its lifetime. Internal and external factors, such as toxic
compounds and radiation, are a threat to the wellbeing of such
organism. Potentially harmful factors comprise factors capable of
distorting cellular processes such as the generation of vital
biomolecules and/or degradation of such molecules, notably nucleic
acids and proteins. For instance, inhibitors of RNA or protein
synthesis, transport or turn-over compromise cellular function.
Additionally, many internal and external factors are capable of
damaging cellular components, such as DNA.
Preservation of an intact genome is of utmost importance to living
cellular organisms. However, the integrity of nucleic acids such as
DNA is continuously challenged. Cells must overcome endogenous (for
instance, metabolic) and exogenous (environmental) threats, as well
as the intrinsic instability of chemical bonds in nucleic acid such
as DNA itself (e.g., deamination and depurination). For instance,
oxidative stress, ultraviolet (UV) light, ionizing radiation (such
as X-rays), and numerous chemicals are capable of inducing a wide
variety of lesions in DNA. An agent capable of inducing a DNA
lesion is called a mutagen. A DNA lesion is defined herein as an
alteration of DNA which involves a change in DNA sequence and/or a
change in DNA structure. A DNA lesion can, for instance, comprise a
DNA (double) strand break and/or an insertion/deletion of at least
one nucleotide.
A DNA lesion can affect cellular processes and can have severe
consequences for the wellbeing of an organism. Direct effects of
DNA lesions at the cellular level comprise inhibition of vital
processes like transcription and replication, triggering cell cycle
arrest. Accumulation of lesions in DNA above certain thresholds can
lead to permanent alterations in the genetic code, replicative
senescence and/or to (programmed) cell death. Permanent alterations
in the genetic code can, for instance, cause changes in metabolic
processes, inborn defects and/or overall functional decline
contributing to (premature) aging. Mutations, specifically in
proto-oncogenes and tumor suppressor genes, are responsible for
tumor initiation and subsequent progression of the multistep
process of carcinogenesis. Replicative senescence and cell death
can enhance the process of aging.
Potential mutagens are often tested with the widely used Ames test.
This test is based upon reversion of mutations in a histidine (his)
operon in the bacterium Salmonella typhimurium. The his operon
encodes enzymes required for the biosynthesis of the amino acid
histidine. Strains with mutations in the his operon are histidine
auxotrophs: they are unable to grow without added histidine.
Revertants that restore the his.sup.+ phenotype will grow on
minimal medium plates without histidine.
In the Ames test, the his.sup.- mutants are mixed with a potential
mutagen and then plated on minimal medium with a very small amount
of histidine. The concentration of histidine used is limiting, so
after the cells go through several cell divisions, the histidine is
used up and the auxotrophs stop growing. However, if the potential
mutagen induces his.sup.+ revertants during the initial few cell
divisions, then each of the resulting revertants will continue to
divide and form a colony. The number of colonies produced is
proportional to how efficiently a mutagen reverts the original
his.sup.- mutation.
A disadvantage of the Ames test is that it is unable to detect
mutagenic agents that are activated by the eukaryotic (organ or
tissue-specific) cellular metabolism (such as the class of p450
enzymes). Although preincubation of the agent to be tested with
cellular extracts may partly overcome this limitation, the assay is
still unreliable as it utilizes a bacterium to predict effects in a
very different organism, such as a mammal, and/or in specific
organs or tissues. Next to entire classes of false negative
outcomes, also a significant number of false positive results have
been obtained with the heterologous procaryotic system. Moreover,
the test detects only mutagenic compounds but does not detect
agents that have mainly a cytotoxic effect or induce deletions, or
other chromosomal aberrations. Finally, the Ames test takes
overnight incubation until the result is obtained. This is due to
the fact that bacterial growth needs to be awaited.
DISCLOSURE OF THE INVENTION
The invention provides a novel test for detecting cells with DNA
lesions. The invention also provides a novel test for detecting
agents that are harmful to eukaryotic organisms. More specifically,
the present invention provides a novel test for detecting agents
that are mutagenic and/or cytotoxic for eukaryotic cells and a
novel test for detecting agents capable of at least in part
inhibiting proteolysis. The invention further provides a method for
determining whether a cell has an impaired DNA damage repair
mechanism.
The invention provides a method for detecting a DNA lesion in a
eukaryotic cell, comprising determining whether an HR23
protein-binding molecule accumulates within the cell. The invention
furthermore provides a method for determining whether an agent is
capable of inducing a DNA lesion in a eukaryotic cell,
comprising:
exposing at least one eukaryotic cell to the agent; and
determining whether an HR23 protein-binding molecule accumulates
within the cell.
Preferably, it is determined whether the HR23 protein-binding
molecule accumulates in the nucleus of the cell.
By a "HR23 protein-binding molecule" is meant herein a molecule,
for instance a peptide or protein, capable of specifically binding
an HR23 protein. The HR23 protein-binding molecule may be a natural
ligand of HR23. Alternatively, the HR23 protein-binding molecule
may be an artificial binding partner of HR23.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1E. Targeted disruption of the mHR23A gene by homologous
recombination.
FIG. (1A) Genomic organization and disruption strategy for mHR23A
depicting the gene, the targeting construct, and the targeted
mHR23A allele. Exons III-VI (and part of exons II and VII) were
replaced by the dominant selectable neomycin resistance marker
transcribed in antisense orientation.
FIG. (1B) Southern blot analysis of BamHI-digested DNA from ES
cells showing the 5.0 kb and 3.5 kb fragment representing the
wild-type and the targeted allele of mHR23A, respectively.
FIG. (1C) Southern blot analysis of BamHI-digested tail DNA from
mHR23A.sup.+/+, mHR23A.sup.+/-, and mHR23A.sup.-/- mice.
FIG. (1D) RNA blot analysis of mHR23A mRNA from mHR23A.sup.+/+,
mHR23A.sup.+/-, and mHR23A.sup.-/- MEFs using mHR23A cDNA as a
probe (upper panel). As a loading control for the amount of RNA,
the blot was reprobed with .beta.-actin cDNA (lower panel).
FIG. (1E) Immunoblot analysis of mHR23A protein in cellular
extracts from mHR23A.sup.+/+, mHR23A.sup.+/-, and mHR23A.sup.-/-
MEFs loaded in equal amounts. Polyclonal antibodies against the
human HR23A protein (upper panel) and the human XPC protein (lower
panel) were utilized. The asterisk indicates an aspecific
cross-reacting band.
FIGS. 2A-2F. Repair characteristics of mHR23A.sup.-/- E13.5 and DKO
E8.5 MEFs.
FIG. (2A) UV survival curves of primary mHR23A.sup.+/+,
mHR23A.sup.+/-, and mHR23A.sup.-/- E13.5 MEFs. XPC.sup.-/-
fibroblasts were included as a negative control. Cells were exposed
to different doses of UV (254 nm). After 4-5 days, the number of
proliferating cells was estimated from the amount of radioactivity
incorporated during a 3 hr pulse with [.sup.3H]thymidine. For each
genotype, identical results were obtained with three other cell
lines (data not shown).
FIG. (2B) Global genome repair (UDS) in primary mHR23A.sup.+/+,
mHR23A.sup.+/-, and mHR23A.sup.-/- E13.5 MEFs. Cells were
irradiated with 16 J/m.sup.2 UV (254 nm) and labeled with
[.sup.3H]thymidine. Incorporation of radioactivity was measured by
autoradiography and grain counting (average of 50 nuclei per cell
line; the standard error of the mean is indicated). XPA.sup.-/-
fibroblasts were measured as negative control. For each genotype,
consistent results were obtained with three other independent cell
lines (data not shown).
FIG. (2C) RNA synthesis recovery (RRS) after UV exposure of primary
mHR23A.sup.+/+, mHR23A.sup.+/- and mHR23A.sup.-/- E13.5 MEFs. Cells
were UV irradiated (10 J/m.sup.2, 254 nm) and allowed to recover
for 16 hours. After a 1 hr pulse labeling with [.sup.3H]uridine,
cells were processed for autoradiography. The relative rate of RNA
synthesis was expressed as the quotient of the number of
autoradiographic grains over the UV-exposed nuclei and the number
of grains over the nuclei of non-irradiated cells (average of 50
nuclei per cell line; the standard error of the mean is indicated).
CSB.sup.-/- cells were used as a negative control. For each
genotype, three other independent lines were assayed with similar
outcome (data not shown).
FIG. (2D) UV survival of E8.5 MEF lines of wild-type, XPC.sup.-/-,
mHR23A.sup.-/-/B.sup.+/-, mHR23A.sup.+/-/B.sup.-/-, and
mHR23A.sup.-/-/B.sup.-/- (DKO).
FIG. (2E) UV-induced UDS in wild-type, XPC.sup.-/- and DKO E8.5
MEFs.
FIG. (2F) RNA synthesis recovery after UV irradiation of wild-type,
XPC.sup.-/- and DKO E8.5 MEFs. For details for panels D-F see
legends to panels A-C respectively and Experimental Procedures. Two
independent experiments using two other DKO cell lines (before the
cultures extinguished, not shown) showed a similar effect on UDS
and RNA synthesis recovery.
FIGS. 3A-3C. XPC expression in DKO E8.5 MEFs.
FIG. (3A) Phase contrast (left panels) and epifluorescence (middle
and right panels) images of fixed wild-type (WT, labeled with latex
beads), XPC.sup.-/- (XPC) and mHR23A.sup.-/-/B.sup.-/- (DKO) MEFs.
Cells were fixed by paraformaldehyde, permeabilized by 0.1% triton
X-100, and subsequently immunolabeled with affinity-purified
polyclonal antibodies against the human XPC protein (middle panels;
stained with goat anti-rabbit Alexa 488-labeled secondary
antibody). Monoclonal antibodies recognizing p62 subunit of TFIIH
(right panels; stained with goat anti-mouse Cy3-labeled secondary
antibody) were used as an internal control. All images were taken
at the same magnification.
FIGS. (3B-C) Immunoblot analysis of XPC protein in cellular
extracts from wild-type, XPC.sup.-/- and DKO E8.5 MEFs using
polyclonal anti-human XPC antibodies (B). Monoclonal anti-p62
antibodies were used as an internal reference for the amount of
protein in each lane (C).
FIGS. 4A-4F. Characterization of DKO cells expressing hHR23B and
XPC-GFP.
(4A) UV survival of wild-type, XPC.sup.-/-, DKO, and DKO MEFs
cotransfected with: hHR23B (h23B), human XPC-GFP (hXPC), and h23B
and hXPC-GFP cDNAs. Cells were exposed to different doses of UV
(254 nm). After 4-5 days, the number of proliferating cells was
estimated from the amount of radioactivity incorporated during a 3
hr pulse with [.sup.3H]thymidine. For details see Experimental
Procedures. For each cDNA construct, similar results were obtained
with at least two other independent stably transfected cell lines
(data not shown).
FIG. (4B) Schematic representation of XPC-EGFP-His6HA-N3 fusion
protein (1208 aa). Indicated are the human XPC protein (940 aa),
the enhanced green fluorescent protein tag (EGFP; 238 aa), and the
hexameric histidine (SEQ ID NO. 21)-hemagglutinin double epitope
tag (His6HA; 17 aa).
FIG. (4C) Immunoblot analysis of XPC expression in cellular
extracts of WT (lane 1), XPC (lane 2), DKO (lane 3), and DKO MEFs
cotransfected with: h23B (lane 4), hXPC-GFP (lane 5), and h23B and
hXPC-GFP (lane 6) cDNAs, using a polyclonal antibody against the
C-terminus of human XPC (upper panel). Monoclonal anti-p62
antibodies were used as a loading control (lower panel).
FIG. (4D) Phase contrast (left) and epifluorescence (right) images
of fixed WT (labeled with latex beads) and DKO cells cotransfected
with hHR23B cDNA. Cells were fixed by paraformaldehyde, followed by
0.1% triton X-100 permeabilization and subsequently immunolabeled
with affinity-purified polyclonal anti-human XPC (right; stained
with goat anti-rabbit Alexa 488-labeled secondary antibody).
Monoclonal anti-p62 antiserum was used as an internal control
(stained with goat anti-mouse Cy3-labeled secondary antibody; data
not shown). Images were taken at the same magnification. Similar
results were obtained with DKO cells cotransfected with hXPC-GFP,
and hHR23B and hXPC-GFP cDNAs (not shown).
FIGS. (4E-4F) Phase contrast (left panels) and epifluorescence
(right panels) images of living DKO cells cotransfected with:
hXPC-GFP (E), or hHR23B and hXPC-GFP (F) cDNAs. All images were
taken at the same magnification. Shown are the nucleus of a first
cell (numbered 1) and the nucleus of a second cell (numbered
2).
FIGS. 5A-5E. Effect of UV, NA-AAF, and proteasome inhibitor on
hHR23B-dependent XPC-GFP level in living DKO cells.
FIG. (5A) Kinetic analysis of living DKO cells expressing
XPC-GFP/hHR23B upon 10 J/m.sup.2 UV-C in time over a period of 30
hours. Percentage XPC-GFP: the percentage of GFP-expressing
fluorescent cells of the total number of cells.
FIG. (5B) Immunoblot analysis of DKO cells expressing
XPC-GFP/hHR23B before exposure to damaging agent (lane 1), 6 hr
after exposure to 10 J/m.sup.2 UV-C (lane 2), and 6 hr after
treatment with 10 .mu.M CBZ-LLL (lane 3) using monoclonal
antibodies recognizing the HA epitope of XPC-GFP (upper panel). A
monoclonal antibody against the p62 subunit of TFIIH (lower panel)
was used as a loading control. A similar outcome was obtained with
two other independent DKO cell lines expressing XPC-GFP/hHR23B
(data not shown).
FIG. (5C) Combined phase contrast and fluorescence images (upper
panels), and epifluorescence images (lower panels) of the same
living DKO cells expressing XPC-GFP/hHR23B before UV (left panels)
and 6 hr after 10 J/m.sup.2 UV-C (right panels). White arrows
indicate the scratch mark on glass coverslips. Numbers represent
the same living cells before and after UV exposure. Identical
results were obtained with two other independent DKO cell lines
expressing XPC-GFP/hHR23B (data not shown). All images were taken
at the same magnification. Shown are the nuclei of three DKO cells
(numbered 1, 2, and 3).
FIG. (5D) Combined phase contrast and fluorescence images (upper
panels), and epifluorescence images (lower panels) of living DKO
cells expressing XPC-GFP/hHR23B before NA-AAF (left panels) and 8
hr after 50 .mu.M NA-AAF (right panels). White arrows indicate the
scratch on glass coverslips. The numbers represent the
corresponding living cells on coverslips before and after NA-AAF
treatment. Identical results were obtained with two other
independent DKO cell lines expressing XPC-GFP/hHR23B (data not
shown). All images were taken at the same magnification. Shown are
the nuclei of five DKO cells (numbered 1, 2, 3, 4, and 5).
FIG. (5E) Combined phase contrast and fluorescence images (upper
panels), and only epifluorescence images (lower panels) of living
DKO cells expressing XPC-GFP/hHR23B before treatment with
proteasome inhibitor CBZ-LLL (left panels) and 6 hr after 10 .mu.M
CBZ-LLL (right panels). All images were taken at the same
magnification.
FIGS. 6A-6B. Local UV damage induces overall XPC stabilization in
nuclei of DKO cells expressing XPC-GFP/hHR23B.
(FIGS. 6A-B) DKO cells expressing XPC-GFP/hHR23B were exposed to 64
J/m.sup.2 UV-C through 5.0 .mu.m pore filters and fixed 5 min (A)
and 2 hours (B) later with paraformaldehyde. Double
immunofluorescent labeling using antibodies against XPA (A, left
panel; stained with goat anti-rabbit Alexa 488-labeled secondary
antibody) and HA epitope (A, right panel; stained with goat
anti-rat Alexa 594-labeled secondary antibody). DAPI stained (B,
left panel) and epifluorescence images without antibody labeling
(B, right panel). Arrows indicate the site of UV-induced local
damage in the nuclei of DKO cells expressing hXCPC-GFP/hHR23B.
Note: Compare the increased fluorescence signal over the entire
nucleus of damaged cells to the signal of non-damaged nuclei for
overall stabilization of XPC (B).
FIGS. 7A-7B. Enhanced DNA repair correlates with high levels of XPC
in UV-induced UDS in DKO cells expressing XPC-GFP/hHR23B.
FIG. (7A) Histogram of UV-induced UDS in DKO cells expressing
XPC-GFP/hHR23B. Five hours after exposure to 10 J/m.sup.2 UV-C,
cells were subsequently irradiated with 16 J/m.sup.2 UV-C and
labeled with [.sup.3H]thymidine for 1 hr (white columns, mean of
UDS level is 25.+-.SEM 1). In parallel, non-prechallenged cells
only exposed to 16 J/m.sup.2 UV-C were used as controls (black
columns, mean of UDS level is 16.+-.SEM 0.6). Asterisks indicate
the mean values of the UDS levels. Incorporation of radioactivity
was measured by autoradiography and grain counting (130 fixed
squares counted per cell line and each square represented
approximately 50% of the nucleus surface). UV-induced UDS of
wild-type (mean 17.+-.SEM 0.8) fibroblasts were measured as
controls (data not shown).
FIG. (7B) Effect of microinjection of XPC-GFP cDNA on UV-induced
UDS in human wild-type (C5RO) fibroblasts. Shown is a micrograph of
a wild-type homodikaryon (numbered 1) microinjected with XPC-GFP in
one of the nuclei and subjected to UV-induced UDS. Prior to UDS,
fluorescence images were captured (inset in B). The injected cell
has a considerably larger number of grains above its nuclei than
the noninjected, surrounding mononuclear cells (numbered 2).
FIGS. 8A-8D. Evidence for XPC shuttling between nucleus and
cytoplasm.
FIG. (8A) Schematic representation of human XPC protein (940 aa),
indicating three putative leucine-rich nuclear export signals
(NES), three putative nuclear location signals (NLS), an N-terminal
acidic stretch, a central Serine-rich domain, and a C-terminal
HR23-binding region (Uchida et al., 2002). The consensus sequence
for NES is indicated separately. Although originally defined as
leucine-rich, other hydrophobic residues (I, F, V, M) have been
shown to be able to substitute for leucines in functional NES
sequences of various proteins (Mowen and David, 2000; Roth et al.,
1998).
FIG. (8B) Amino acid sequence comparison between mouse and human
XPC NES-like domains. Numbers indicate the location of the amino
acids within the respective proteins. Closer examination of NES2
and NES3 revealed multiple conserved leucine-rich regions.
FIGS. (8C-D) Heterokaryon nuclear-cytoplasmic shuttling assay using
DKO cells expressing XPC-GFP(His.sub.6HA)/hHR23B and HeLa cells.
Six hours prior to cell fusion, cells were exposed to 10 J/m.sup.2
UV-C. After cell fusion, cells were cultured either in the absence
(C) or in the presence (D) of the nuclear export inhibitor LMB (10
ng/ml). Four hours after fusion, cells were fixed and immunostained
with anti-HA (left) to detect the fusion protein and
anti-hERCC1-specific antibody (middle) to recognize HeLa cells. The
right picture is a phase contrast image of the same cells. For
clarity, mouse nuclei were marked by (1) and human (HeLa) nuclei
were marked by (2).
FIG. 9. Model for the DNA damage and HR23-dependent regulation of
XPC and GG-NER. In the total absence of the HR23 proteins
(mHR23A/B-decifient), XPC is intrinsically unstable and targeted
for ubiquitin-dependent proteolysis via the 26S proteasome. In view
of the parallel with p53 nucleo-cytoplasmic shuttling, it was
postulated that XPC is degraded in the cytoplasm. As a consequence,
the steady-state level of XPC is decreased, resulting in reduced
GG-NER capacity (upper panel). Under normal conditions, HR23
proteins (indicated as 23) control XPC degradation leading to
partial stabilization of XPC (in a complex with HR23 and CEN2 (C)).
Higher steady-state levels of XPC result in proficient GG-NER
(middle panel). NER-type DNA damage (e.g., UV irradiation) induces
a further increase in XPC/HR23/CEN2 protein levels through nuclear
retention of XPC bound to lesions, and accordingly enhances GG-NER
capacity (lower panel). A comparable HR23-mediated stabilization
mechanism may hold for other factors and cellular pathways in which
HR23 proteins are implicated (see discussion for further
explanation).
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Preferably, the DNA lesion comprises a lesion that is a substrate
for global genome nucleotide excision repair and/or base excision
repair. More preferably, the HR23 protein-binding molecule
comprises xeroderma pigmentosum group C protein, 3-methyladenine
DNA glycosylase, CREB, p53, or a functional part, derivative and/or
analogue thereof.
Xeroderma pigmentosum group C (XPC) protein is involved in a DNA
repair mechanism called nucleotide excision repair (NER). NER
primarily focuses on helix-distorting injuries, including
UV-induced cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4)
pyrimidone photoproducts (6-4PP), as well as numerous chemical DNA
adducts (Friedberg et al., 1995). Inherited defects in NER are the
cause of several severe diseases, such as the cancer-prone syndrome
xeroderma pigmentosum (XP). Patients are characterized by extreme
sun sensitivity, sun-induced pigmentation anomalies, and a
>2000-fold predisposition to UV-induced skin cancer. Moreover,
an impaired DNA repair mechanism such as NER or BER (described
below) is involved in alteration of cells and their response to
genotoxic agents.
NER entails a multistep reaction and requires the coordinated
action of .about.30 proteins implicated in damage detection, helix
opening, lesion verification, dual incision of the damaged strand
bracketing the injury, removal of the 25-30 base damage-containing
oligonucleotide, gap-filling DNA synthesis and ligation
(Hoeijmakers, 2001). Two NER subpathways exist: global genome NER
(GG-NER), operating genome wide and transcription-coupled repair
(TCR), focusing on transcription-blocking lesions in the
transcribed strand of active genes (Hanawalt, 2000). Most XP genes
are implicated in both NER subpathways, but XPC-deficient cells are
unique in being selectively deficient in GG-NER.
3-Methyladenine DNA glycosylase (MAG) is involved in a DNA repair
mechanism called base excision repair (BER). BER corrects base
alterations induced by endogenous and/or exogenous oxidative
events, ionizing radiation and small alkylating agents.
Examples of potentially mutagenic BER lesions are 8-oxoguanine,
O.sup.6-methylguanine, deaminated methylated cytosine and thymine
glycol. Base excision repair is initiated by MAG and several other
glycosylases. Accumulating evidence implicates unrepaired BER
lesions in the aging of somatic cells.
According to the present invention, a DNA lesion in a eukaryotic
cell results in accumulation of an HR23 protein-binding molecule
within the cell. For instance, an NER- and/or BER-sensitive DNA
lesion results in rapid accumulation of XPC and/or MAG of a
eukaryotic cell, especially in the nucleus. Accumulation of an HR23
protein-binding molecule, such as XPC and/or MAG, is at least in
part due to the fact that the HR23 protein-binding molecule is
stabilized by the chaperone proteins HR23A and/or HR23B if a DNA
lesion is present. Both chaperone proteins are normally present in
eukaryotic cells. In terms of the invention, a "HR23 protein" is
defined as an HR23A or HR23B protein. It has been shown by the
present inventors that only one of them suffices for stabilizing
XPC. If no NER- or BER-sensitive DNA lesion is present, XPC and MAG
are rapidly degraded. A complex comprising HR23 and an HR23
protein-binding molecule is involved in DNA repair. For instance, a
complex comprising XPC and HR23 performs a damage-sensing step
within GG-NER, triggering subsequent association of the
NER-involved proteins TFIIH, XPG, XPA, RPA, and ERCC1/XPF.
Accumulation of XPC, or a functional part, derivative and/or
analogue thereof, is therefore indicative for the presence of a DNA
lesion that is a substrate for GG-NER.
As another example, HR23 is also capable of associating with MAG.
Since MAG plays an important role in an initiation of a BER
response, accumulation of MAG, or a functional part, derivative
and/or analogue thereof, is indicative of the presence of a DNA
lesion that is a substrate for BER.
A number of other binding partners for at least one of the HR23
proteins are found, including cell cycle checkpoint proteins such
as CREB and p53 as well as proteins implicated in mitosis. These
binding partners are also suitable for a method of the
invention.
HR23A and HR23B are homologues of the Saccharomyces cerevisiae gene
RAD23. The present inventors have cloned the two human homologues
of RAD23, designated hHR23A and hHR23B. HR23 proteins contain a
ubiquitin-like (Ubl) N-terminus and two ubiquitin-associated (UBA)
domains pointing to multiple links with the ubiquitin system. The
Ubl domain of yeast RAD23 is important for UV survival and for
interaction with the 26S proteasome, whereas the UBA domains enable
binding to ubiquitin. Until the present invention, the functional
relationship between RAD23, DNA repair and the ubiquitin system was
unclear.
Now that it has been found that HR23 protein-binding proteins such
as XPC and MAG accumulate in a eukaryotic cell upon a DNA lesion, a
rapid and sensitive test is provided. A method of the invention is
preferred over the widely used Ames test, because readout is based
on the detection of accumulation of an HR23 protein-binding
molecule (for instance, by way of fluorescence) and not on
bacterial growth. A method of the invention can be performed
quicker and allows for smaller quantities of a test compound as
compared to the Ames test. Preferably, a method of the invention is
performed within 3 to 6 hours. Moreover, a method of the invention
provides more information about the effect of a test compound upon
a eukaryotic organism as compared to the Ames test. For instance,
information is provided about transport of a test compound into a
eukaryotic cell and into the nucleus of such cell, possible
enzymatic modification of the test compound by a eukaryotic
organism, and susceptibility of eukaryotic DNA. Moreover, a test of
the invention provides information about the type of DNA damage
induced by an agent, since different types of DNA lesions involve
accumulation of specific HR23 protein-binding molecules.
Potentially mutagenic or cytotoxic agents can be tested by exposing
a eukaryotic cell to the agent and determining whether an HR23
protein-binding molecule such as XPC and/or MAG, or a functional
part, derivative and/or analogue thereof, accumulates within the
cell. In terms of the invention, by "an agent capable of inducing a
DNA lesion" is meant an agent, such as for instance a compound or
radiation, which is capable of inducing at least one DNA lesion. By
"an agent capable of inducing a DNA lesion that is a substrate for
GG-NER and/or BER" is meant an agent, such as for instance a
compound or radiation, which is capable of inducing at least one
DNA lesion that is normally recognized by a GG-NER and/or BER
system. By "normally recognized" is meant that in a (preferably
naturally occurring) cell with functioning GG-NER and/or BER
system, such lesion is recognized by the GG-NER and/or BER system.
Of course, a capability of inducing DNA lesions often strongly
depends on the dose of compound/radiation. Therefore, it is often
suitable to test several amounts of test compound or several
intensities of radiation with a method of the invention. However,
if it is only questioned whether a specific dose of
compound/radiation (for instance, present in a new chemical
compound, in a new procedure of purification, or in, for example,
soiled ground) is mutagenic, a test of the invention can be
performed with only that dose.
A eukaryotic cell can be exposed to an agent in many different
ways, which are known in the art. For instance, a potentially
mutagenic compound can be administered to a culture comprising the
eukaryotic cell. Alternatively, the compound can be administered to
a non-human animal comprising the cell. Alternatively, a eukaryotic
cell, for instance, as part of a cell line or as part of a
non-human animal, can be exposed to radiation. In one embodiment,
several different doses of compound/radiation are used.
With a method of the invention, accumulation of an intact XPC
and/or MAG protein in a eukaryotic nucleus can be determined.
Alternatively, accumulation of a functional part, derivative and/or
analogue of XPC and/or MAG can be tested. In a preferred
embodiment, the XPC protein comprises a human XPC protein or a
functional part, derivative and/or analogue thereof.
A functional part, derivative and/or analogue of an XPC and/or MAG
protein can be provided to a eukaryotic cell with conventional
methods known in the art, such as microinjection or transfection
procedures. Such functional part, derivative and/or analogue can be
provided to a eukaryotic cell by use of a nucleic acid encoding the
functional part, derivative and/or analogue. Of course, the nucleic
acid is preferably suitable for expression within the cell. As
shown in the examples, it is also possible to provide a cell with a
nucleic acid encoding a whole XPC and/or MAG protein. The protein
may be an endogenous XPC and/or MAG protein, or may be derived from
a different kind of organism.
In a preferred embodiment, the HR23 protein-binding molecule is
labeled to allow for easy detection. In one preferred embodiment,
the label comprises green fluorescent protein (GFP) or luciferase.
If GFP is used as a label, accumulation of an HR23 protein-binding
molecule such as XPC and/or MAG can be easily detected with a
microscope or a fluorescent activated cell sorter (FACS) for easy
and rapid quantitative readout. An HR23 protein-binding molecule
can be labeled in many different ways and with many different
labels known in the art. For instance, the label may be coupled to
the molecule by way of a (flexible) linker. The linker can be a
peptide.
The label can also be linked to the molecule in the form of a
fusion protein, comprising both the molecule and the label. A
nucleic acid can be constructed encoding such fusion protein by
methods known in the art. Of course, the person skilled in the art
can think of alternative ways of linking a label to XPC and/or MAG,
or to a functional part, derivative and/or analogue thereof.
Besides labeling of an HR23 protein-binding molecule, accumulation
of the molecule can also be detected in different ways that are
known in the art. For instance, an antibody directed towards the
molecule can be used. Binding of the antibody can be detected by
staining the antibody, an affinity column may be used, etc. As
another possibility, the molecule can be rendered radioactive.
A functional part of an XPC and/or MAG protein is defined as a part
which has the same kind of properties as XPC and/or MAG in kind,
not necessarily in amount. A functional part of XPC and/or MAG is,
for instance, also capable of binding to HR23A and/or HR23B, and/or
capable of entering the nucleus of a eukaryotic cell, optionally
when bound to HR23A or HR23B. A functional derivative of an XPC
and/or MAG protein is defined as a protein which has been altered
such that the properties of the molecule are essentially the same
in kind, not necessarily in amount. A derivative can be provided in
many ways, for instance, through conservative amino acid
substitution.
A person skilled in the art is well able to generate analogous
compounds of an XPC and/or MAG protein. This can, for instance, be
done through screening of a peptide library. Such analogue has
essentially the same properties of an XPC and/or MAG protein in
kind, not necessarily in amount.
As used herein, "an XPC protein" and "XPC" are used interchangeably
and can also mean a functional part, derivative and/or analogue of
an XPC protein. Likewise, "a MAG protein" and "MAG" are used
interchangeably herein and can also mean a functional part,
derivative and/or analogue of a MAG protein.
In a preferred embodiment, a method of the invention is provided
wherein the eukaryotic cell is overexpressing HR23A and/or HR23B
protein, or a functional part, derivative and/or analogue thereof.
As shown in the examples, very good results are obtained if HR23A
and/or HR23B is overexpressed. Overexpression of HR23A and/or HR23B
can be performed in different ways. For instance, a nucleic acid
encoding HR23A and/or HR23B can be constructed, preferably with a
strong promoter. Such nucleic acid may comprise several copies of a
gene encoding HR23A and/or HR23B. Overexpression of HR23A and/or
HR23B can be induced by administration of the nucleic acid to a
cell capable of expressing the nucleic acid. In one embodiment, the
nucleic acid encodes human HR23A and/or human HR23B protein or a
functional part, derivative and/or analogue thereof. In another
embodiment, the nucleic acid encodes murine HR23A and/or murine
HR23B protein or a functional part, derivative and/or analogue
thereof.
The nucleic acid may be expressed in a eukaryotic cell in addition
to endogenously expressed HR23A and/or HR23B. Alternatively, the
cell may be rendered deficient of endogenous HR23A and/or HR23B.
The nucleic acid preferably comprises a stronger promoter than the
endogenous genes of HR23A and/or HR23B, enabling the cell to
overexpress HR23A and/or HR23B.
In one embodiment, a method of the invention is provided wherein
the eukaryotic cell is a mammalian cell. Preferably, the cell is a
murine cell, more preferably a mouse embryonic fibroblast. The cell
can be part of a cell line, such as a mouse embryonic fibroblast
cell line. According to one embodiment, the cell is deficient in
endogenous HR23A and/or HR23B protein. The cells of the cell line
are also preferably deficient in endogenous HR23A and/or HR23B
protein. Murine HR23A or HR23B protein is called mHR23A or mHR23B.
A cell which is deficient for both endogenous HR23A and HR23B
protein is preferably artificially provided with HR23A and/or
HR23B. As disclosed in the examples, only one kind of HR23 protein
is sufficient to preserve GG-NER activity. In another preferred
embodiment, a method of the invention is provided wherein the XPC
protein or a functional part, derivative and/or analogue thereof
comprises a human XPC protein or a functional part, derivative
and/or analogue thereof.
The invention also provides a method for screening of agents
capable of at least in part inhibiting a cellular process that
normally results in accumulation of HR23 protein-binding molecules.
For instance, in response to a DNA lesion, an HR23 protein-binding
molecule accumulates within a cell. This involves many cellular
processes, such as RNA synthesis, RNA processing, RNA transport,
and/or RNA translation. If an agent is capable of inhibiting such
process, accumulation of an HR23 protein-binding molecule will not
occur or will occur less when a DNA lesion is present. The
invention therefore provides a method for determining whether an
agent is capable of at least in part inhibiting a cellular process,
such as proteolysis, nucleo-cytoplasma shuttling, RNA synthesis,
RNA processing, RNA transport, and/or RNA translation, the process
resulting in accumulation of HR23 protein-binding molecule within a
cell, comprising: exposing at least one eukaryotic cell to the
agent; and determining whether an HR23 protein-binding molecule
accumulates therewithin. Preferably, the eukaryotic cell comprises
a DNA lesion.
In a preferred embodiment, the HR23 protein-binding molecule
comprises xeroderma pigmentosum group C protein, 3-methyladenine
DNA glycosylase, CREB, p53, or a functional part, derivative and/or
analogue thereof.
Preferably, the proteolysis comprises proteasomal proteolysis. It
has been shown by the present inventors that hXPC-GFP is mainly
degraded via ubiquitin/proteasome-dependent proteolysis.
In one aspect, the invention provides a mammalian cell which is
deficient in endogenous HR23A protein or endogenous HR23B protein.
In one embodiment, the cell comprises a murine cell, preferably a
mouse embryonic fibroblast. With a cell of the invention, a cell
line can be generated. Such cell line is suitable for high
throughput tests of compounds, for instance, potentially mutagenic
or cytotoxic compounds or compounds potentially capable of
inhibiting proteolysis, with a method of the invention.
A cell line comprising a cell of the invention is therefore also
herewith provided.
As outlined in the examples, a non-human animal comprising a cell
of the invention is also very suitable for testing and
investigation purposes. The invention therefore also provides a
non-human eukaryotic organism which is deficient in endogenous
HR23A protein and/or endogenous HR23B protein. Since total HR23
deficiency is incompatible with animal life, an animal of the
invention should comprise HR23, either one of the endogenous HR23A
or HR23B proteins or a functional part, derivative and/or analogue
thereof, or an exogenous (such as human or murine) HR23 protein or
a functional part, derivative and/or analogue thereof. In one
embodiment, the animal is provided with an exogenous controllable
HR23 transgene. The invention also provides a non-human animal of
the invention with compromised (endogenous) HR23 functions in a
conditional fashion.
According to the invention, an HR23 protein-binding molecule
accumulates in a cell in response to a DNA lesion. For instance,
XPC and/or MAG accumulate(s) in a eukaryotic cell in response to a
DNA lesion that is a substrate for GG-NER and/or BER. This applies
to cells with a functioning GG-NER and/or BER system. However, if a
cell's GG-NER and/or BER system is essentially impaired, XPC and/or
MAG will not accumulate in the nucleus in response to DNA damage.
Hence, accumulation of an HR23 protein-binding molecule in a cell
that is exposed to a DNA-affecting agent is indicative of an
essentially functioning DNA repair system. The invention therefore
provides a method for determining whether a cell has an at least
partly impaired DNA repair system, comprising: exposing the cell to
an agent capable of inducing a DNA lesion; and determining whether
an HR23 protein-binding molecule accumulates within the cell.
Preferably, the HR23 protein-binding molecule comprises xeroderma
pigmentosum group C protein and/or 3-methyladenine DNA glycosylase,
or a functional part, derivative and/or analogue thereof.
Impaired NER and/or BER activity is associated with severe
disorders, such as xeroderma pigmentosum (XP), cockayne syndrome
(CS) and trichothiodystrophy (TTD).
Xeroderma pigmentosum is due to a mutation in one of seven genes
involved with NER (designated XPA to XPG). Parchment skin
(xeroderma) and freckles (pigmentosum) are the prominent cutaneous
hallmarks of XP patients. These manifestations are strikingly
restricted to sun-exposed areas of their skin. Typically, sun
exposure of XP patients causes a progressive degenerative
alteration of the skin and eyes, beginning as early as the age of 2
years. Furthermore, XP is associated with an elevated frequency
(>1000-fold) of sunlight-induced skin cancers, which are also
largely confined to sun-exposed areas like the face, neck, head and
even the tip of the tongue. XP patients mainly develop basal cell
or squamous cell carcinomas, seen in at least 45% of all XP
patients, many of whom often have multiple primary neoplasms, and
less frequently melanomas (5% of patients). The mean age of onset
for skin neoplasms is 8 years, which is about 50 years earlier than
in the general population. The main cause of death in XP
individuals is neoplasia, which reduces the lifespan by
approximately 30 years. XP patients also have a 10- to 20-fold
increased risk of developing several types of internal cancers
before the age of 20 years. Abnormalities in the immune system
detected in XP patients are likely to contribute to the development
of (skin) tumors.
A fraction of XP patients (.about.18%) displays progressive
neurologic degeneration secondary to a loss of neurons. This
feature seems to be related to the significance of the NER defect.
For example, XPC patients, who only have the GG-NER defect, usually
do not develop neurologic abnormalities, and if so, symptoms appear
much later in life compared to TC-NER-defective XPD and completely
NER-deficient XPA patients. A possible explanation for the onset of
neurologic abnormalities in XP individuals is that defective DNA
repair of endogenous, oxidative NER lesions in neurons triggers
cell death.
The genetic heterogeneity of XP patients is accompanied by
heterogeneity in severity of the repair defect and of the
consequent symptoms. The most severely affected patients are XPA,
XPB, XPD and XPG individuals. The two most common forms of XP are
XPA and XPC. The group of XPD patients is the most heterogeneous,
with a level of residual repair synthesis between 15 and >50%.
Furthermore, XPF patients are moderately UV sensitive and show
intermediate repair synthesis, indicative of mutations that lead to
poor but not complete abolishment of NER. This could be due to the
anticipated dual function of the ERCC1-XPF complex in NER and
recombination repair. A null allele for ERCC1 or XPF and the
consequential defect of cross-link repair are predicted to be
incompatible with life. All XP patients of complementation groups A
to G are defective in both NER subpathways, with the exception of
XPC and XPE whose NER defect is limited to GG-NER. The
susceptibility to sunburn of XPC patients is no different from
normal individuals, indicating that TC-NER alone is sufficient to
prevent this acute response to UV exposure. XPC cells have a
residual UDS level of 15-30% due to functional TC-NER and are
therefore less sensitive to UV than XPA or XPD cells. Patients in
the XP-variant group have mild to severe skin symptoms and usually
display a normal functioning central nervous system. Unlike
classical XP, XPV patients show a normal level of NER activity but
lack the capacity to efficiently replicate damaged DNA, leading to
error-prone replication and a hypermutable phenotype. This
phenotype, together with the increased frequencies of genomic
rearrangements observed in XPV cells, may cause the elevated
sun-induced carcinogenesis seen in these patients.
Cockayne Syndrome (CS)
CS is a very pleiotropic disorder characterized by cutaneous
photosensitivity (with or without thin or dry skin and hair),
severe postnatal growth failure (cachectic dwarfism), mental
retardation, and progressive neurologic dysfunction. CS cells are
sensitive to a number of DNA-damaging agents (including UV) due to
a defect in TC-NER. In contrast to patients suffering from the
prototype NER-deficient disorder XP, CS individuals are not
predisposed to skin cancer. Other common CS symptoms include
sensorineural hearing loss, progressive ocular abnormalities (such
as pigmentary retinopathy and/or cataracts), wizened bird-like
faces, impaired sexual development, skeletal abnormalities
(typically resulting in short stature), dental caries, kyphosis
(hunchback), and premature osteoporosis (demineralization). The
progressive neurological degeneration has a very early onset in CS
individuals (beginning around 2 years of age) and is caused by
dysmyelination. The mean age of death in CS is 12.5 years and
mainly secondary to pneumonia, which in turn could be due to the
generally poor condition of the patients. Clearly, CS clinical
symptoms are much more severe than the classical XP condition and
go beyond photosensitivity. Photosensitivity and other XP-like
features (such as pigmentation abnormalities and predisposition to
skin cancer) can be attributed to the NER defect. However, the
severe development and neurological manifestations of CS cannot be
explained by NER. The transcriptional engagement of CSA and CSB
(analogous to XPB and XPD) suggests that transcription deficiency,
perhaps induced by DNA damage, also contributes to the clinical
pictures. In some cases, CS features are found in combination with
XP, due to specific mutations in the XPB, XPD or XPG genes. Cells
from CSA, CSB and XPG individuals with severe CS symptoms are
slightly sensitive to ionizing radiation in addition to UV light.
It is hypothesized that inefficient TCR of oxidative lesions (e.g.,
thymine glycol) which block transcription underlies this ionizing
radiation sensitivity although ionizing radiation is a poor
inhibition of transcription in general. This indicates an
additional role of CSA, CSB and XPG in coupling arrested
transcription with both BER and NER, and suggests a general
repair-transcription coupling deficiency as the major cause of the
extensive variations in symptoms and severity of the CS phenotype.
The developmental defects and the premature aging-related symptoms
of CS can be attributed to the incomplete repair of endogenous
oxidative damage, which in turn causes cellular malfunction and/or
induction of apoptosis. The defective TCR in CS cells enhances
their p53-dependent apoptotic response, contributing to the
elimination of cells that potentially carry oncogenic mutations.
This explains the lack of cancer predisposition in CS after UV
exposure. Numerous other CS-like patients have been identified, for
example, CAMFAK (for cataracts, microcephaly, failure to thrive,
kyphoscoliosis) and COFS (cerebro-oculofacial syndrome), but these
patients fail to exhibit pronounced photosensitivity in spite of
the fact that cells of the patients display defective recovery of
RNA synthesis, suggesting the possibility of a partial
transcription defect without the accompanying TC-NER defect of
CS.
Trichothiodystrophy (TTD)
TTD is caused by neurectodermal dysplasia which causes a collection
of symptoms referred to by the acronym PIBIDS: photosensitivity,
ichthyosis, brittle hairs, intellectual impairment, decreased
fertility, and short stature.
Skeletal abnormalities are also frequently observed, including a
peculiar bird-like face, a receding chin, and retardation of
skeletal age. Moreover, axial osteoschlerosis (abnormal hardening
of the bone), peripheral osteoporosis and kyphosis have been
reported. The striking ectodermal symptoms (brittle hair and
dystrophic nails) are unique for TTD. However, the remainder of the
clinical features are strikingly similar to CS, including the
absence of cancer predisposition. The photosensitivity in TTD
patients is due to a defect in NER caused by a mutation in one of
three genes: XPB, XPD or TTDA. The NER defect in all but two of 20
studied UV-sensitive TTD families can be assigned to the XPD
complementation group. Despite the NER defect, the pigmentation
abnormalities are relatively mild compared to classical XP. The
typical brittleness of TTD hair is caused by a substantial
reduction in the content of hair-specific cysteine-rich matrix
proteins that provide the hair shaft with its natural strength by
cross-linking the keratin filaments. Growth retardation (cachectic
dwarfism) in TTD patients is a very heterogeneous clinical symptom
and--when severe--can be associated with death in early childhood.
TTD, like CS, is considered to be a repair/transcription syndrome.
Mutations in XPD may not only affect the NER function but also
cripple transcription by TFIIH, accounting for the typical TTD and
CS phenotypes. Consistent with this idea, all causative mutations
in XPD have been found to be disease-specific. Recently, the
phenotype of two unrelated TTDA patients was directly attributed to
a limiting amount of TFIIH, probably secondary to a mutation in a
gene determining the complex stability. A reduced TFIIH level has
an effect on its repair function and also on its role in basal
transcription.
A method of the invention is particularly suitable for determining
whether an individual suffers from, or is at risk of suffering
from, a disease associated with impaired DNA repair activity, such
as XP, CS and/or TTD. With a method of the invention it can, for
instance, be determined whether a cell from the individual has an
at least partly impaired GG-NER and/or BER system. If the cell
appears to have an impaired DNA repair system, it is indicative for
disease.
In one embodiment, the invention therefore provides a method for
determining whether an individual suffers from, or is at risk of
suffering from, a disease related to an at least partly impaired
DNA repair system, comprising: obtaining at least one cell from the
individual; exposing the cell to an agent capable of inducing a DNA
lesion; and determining whether an HR23 protein-binding molecule
accumulates within the cell. Preferably, the HR23 protein-binding
molecule comprises xeroderma pigmentosum group C protein and/or
3-methyladenine DNA glycosylase, or a functional part, derivative
and/or analogue thereof.
In a preferred embodiment, the disease comprises xeroderma
pigmentosum, cockayne syndrome, and/or trichothiodystrophy.
A kit of parts comprising a cell and/or a cell line of the
invention is also herewith provided. Preferably, a kit of parts of
the invention comprises an agent capable of inducing a DNA lesion,
such as a lesion that is a substrate for global genome nucleotide
excision repair and/or base excision repair. More preferably, the
kit of parts further comprises a detection system for detecting a
change in level of an HR23 protein-binding molecule. Most
preferably, the kit of parts comprises a detection system for
detecting a change in level of XPC, MAG, CREB, p53, or a functional
part, derivative and/or analogue thereof. A kit of parts of the
invention is particularly suitable for performing a method of the
invention.
According to the invention, XPC, MAG and HR23 play an important
role in the GG-NER and BER systems. Deficiency of at least one of
these proteins is, therefore, challenging for an organism's NER
and/or BER system. Such deficiencies can, at least partly, be
overcome by providing at least one cell from an individual with at
least one of the proteins. Likewise, other DNA repair deficiencies
can be overcome by providing at least one cell from an individual
with at least one HR23 protein-binding molecule. Preferably, the
protein is provided by gene therapy. The invention thus provides a
method for treating a disease related to an at least partly
impaired DNA repair system, comprising: providing at least one cell
of an individual suffering from, or at risk of suffering from, the
disease with a nucleic acid molecule encoding HR23 protein and/or
an HR23 protein-binding molecule, or a functional part, derivative
and/or analogue thereof. Preferably, the HR23 protein-binding
molecule comprises XPC, MAG, CREB, p53, or a functional part,
derivative and/or analogue thereof. More preferably, the DNA repair
system comprises a global genome nucleotide excision repair system
and/or base excision repair system.
Multiple engagements between HR23 and cell cycle regulation are
apparent. Since HR23 is capable of binding to primary damage
sensors such as XPC and MAG, HR23 can be used to influence
coordinated control of major cellular DNA damage response pathways,
including DNA repair, cell cycle progression and checkpoints,
apoptosis and chromosome segregation. In one aspect, the invention
therefore provides a use of an HR23 protein, or a functional part,
derivative and/or analogue thereof, for influencing apoptosis, cell
cycle control and/or chromosome segregation in a eukaryotic cell.
Such assays are very relevant for testing the action of novel
therapeutic agents for their mutagenic and/or cytotoxic properties,
and for detection of side effects of specific treatments and/or
medication.
The invention is further explained in the following examples. The
examples only serve to clarify the invention and do not limit the
scope of the invention in any way.
EXAMPLES
Experimental Procedures
Construction of mHR23A Targeting Vector
An Ola129 mHR23A targeting construct was generated by converting
the BglII site in exon II of clone pG7M23Ag1 (containing a 4 kb
genomic EcoRI fragment subcloned in pGEM7) into a ClaI site, which
(due to a ClaI site in the polylinker) allowed deletion of
sequences downstream of the BglII site in exon II (clone
pG7M23Ag7). Next, the remaining EcoRI site was removed by filling
in the overhangs with Klenow, resulting in clone pG7M23Ag9. After
changing the BstXI site into an SalI site, the 3 kb XhoI-SalI
fragment was cloned into SalI-digested pGEM5, resulting in clone
pG5M23Ag17. Next, the 3' arm of the construct, consisting of a
Klenow-blunted 1.5 kb SmaI-XbaI fragment starting at the SmaI site
in exon VII, was inserted in the blunted NdeI site of pG5M23Ag17
(giving pG5M23Ag20), followed by insertion of a Neo marker cassette
in antisense orientation in the ClaI site (giving pG5M23Ag24).
Finally, the NotI-NsiI insert of pG5M23Ag24 was recloned into a
pGEM-9Zf(-) based vector containing a 2.8 kb thymidine kinase (TK)
marker cassette (giving pG5M23Ag30).
ES Cell Culture and Transfection
The Ola129-derived ES cell line E14 was electroporated with the
mHR23A targeting construct and cultured on dishes treated with
gelatin as described previously (Ng et al., 2002). G418
(GENETICIN.RTM., Gibco, final concentration 200 .mu.g/ml) was added
24 hr after electroporation and cells were maintained under
selection for 6-8 days. Genomic DNA from G418-resistant clones was
digested with BamHI and subjected to Southern blot analysis using a
0.6 kb XbaI-RsaI fragment (3' external to the construct) as a
probe. Targeted clones were subsequently screened with a Neo cDNA
probe (ClaI fragment) to confirm proper homologous recombination in
the 5' arm.
Generation of the mHR23A.sup.-/- and mHR23A.sup.-/-/B.sup.-/- (DKO)
Mice and Fibroblasts
Cells from two independent targeted clones with 40 chromosomes were
injected into 3.5-day-old blastocysts isolated from pregnant
C57BL/6 females (Ng et al., 2002). Male chimeric mice were mated
with C57BL/6 females to obtain heterozygous animals. Germ line
transmission was observed in the coat color of F1 offspring.
Heterozygous males and females for mHR23A were interbred to
generate mHR23A.sup.+/+, mHR23A.sup.+/-, and mHR23A.sup.-/- mice.
For the generation of double mutant mHR23A/B mice, male and female
animals heterozygous for both mHR23A and mHR23B (Ng et al., 2002)
were interbred. Genotyping was performed by Southern blot or PCR
analysis of genomic DNA from tail biopsies of 10-14 day old
pups.
Primary mHR23A MEFs (three independent lines per genotype) were
isolated from day 13.5 embryos (E13.5) obtained from matings
between mHR23A.sup.+/- mice. Double mutant mHR23A/B MEFs were
isolated from day 8.5 embryos (E8.5) derived from different
crossings between mHR23A.sup.+/-/B.sup.+/- and
mHR23A.sup.-/-/B.sup.+/- mice. Part of the embryo was used for
genotyping and the remaining tissue was minced and immersed in a
thin layer of F10/DMEM culture medium (Gibco BRL) supplemented with
15% fetal calf serum, 2 mM glutamate, and 50 .mu.g/ml penicillin
and streptomycin. Spontaneously immortalized (established) cell
lines were obtained by continuous subculturing of primary MEFs.
For the genotyping of E8.5 embryos, the yolk sac was used as
described (Gurtner et al., 1995). In short, the yolk sac was
collected in 20 .mu.l of water and immediately frozen in dry ice.
Samples were heated for 5 min at 95.degree. C. and incubated with 1
.mu.l of proteinase K (10 mg/ml) for 1 hr at 55.degree. C.
Proteinase K was heat-inactivated for 5 min at 95.degree. C. PCR
analysis was performed using the three primer sets described below
for 30 cycles (93.degree. C., 1 min; 55.degree. C., 1 min;
72.degree. C., 90 sec) using mHR23A and mHR23B primers.
Primer set 1: mHR23Ap1 (5'-atg-gga-ctt-ggg-cat-agg-tga-3') (SEQ ID
NO:1), mHR23Ap2 (5'-tct-tca-gcc-agg-cct-ctt-ac-3') (SEQ ID NO:2)
and anti-sense neo (5'-atc-tgc-gtg-ttc-gaa-ttc-gcc-aat-g-3') (SEQ
ID NO:3) giving 243 and 350 by PCR fragments from the wild-type and
targeted allele, respectively. Primer set 2: mHR23Bp1
(5'-gta-aag-gca-ttg-aaa-gag-aag-3') (SEQ ID NO:4), mHR23Bp2
(5'-cta-cag-tct-tgt-ttc-tga-cag-3') (SEQ ID NO:5) and anti-sense
pgk3 (5'-tag-ggg-agg-agt-aga-agg-tg-3') (SEQ ID NO:6) giving 202
and 600 bp PCR fragments from the wild-type and targeted allele,
respectively.
DNA Repair Assays and Microneedle Injection
UV sensitivity was determined as described (Ng et al., 2002). MEFs
cultures were exposed to different doses of UV-C light (254 nm,
Philips TUV lamp) and allowed to grow for another 3-5 days before
reaching confluence. The number of proliferating cells was
estimated by scintillation counting of the radioactivity
incorporated during a 3 hr pulse with [.sup.3H]thymidine (5
.mu.Ci/ml, specific activity (s.a.): 50 Ci/mmole; Amersham). Cell
survival was expressed as the ratio of .sup.3H incorporation in
irradiated and non-irradiated cells.
UV-induced global genome repair was assayed using the UDS method as
described (Vermeulen et al., 1994). Cells were exposed to 16
J/m.sup.2 of 254 nm UV light and labeled with [methyl-3H]thymidine
(10 .mu.Ci/ml, s.a.: 50 Ci/mmole). Repair capacity was quantified
by grain counting after autoradiography.
RNA synthesis recovery after UV irradiation was measured according
to Ng et al. (2002). Cells were exposed to 10 J/m.sup.2 of 254 nm
UV light, allowed to recover for 16 hr, labeled with
[5,6-3H]uridine (10 .mu.Ci/ml, s.a.: 50 Ci/mmole), and processed
for autoradiography. The relative rate of RNA synthesis was
expressed as the number of autoradiographic grains over the
UV-exposed nuclei divided by the number of grains over the nuclei
of non-irradiated cells on parallel slides.
Microneedle injection of control cells (C5RO) was performed as
described previously (Vermeulen et al., 1994). After injection of
at least 50 homopolykaryons, cells were cultured for the desired
time in normal culture medium before they were assayed for their
repair capacity by means of UV-induced UDS.
RNA and Protein Analysis
Total RNA was isolated from mHR23A MEFs using an RNEASY.RTM. Mini
Kit (Qiagen). 20 .mu.g of total RNA was separated on a 0.9% agarose
gel and transferred to HYBOND.RTM.-N+ membrane (Amersham Pharmacia
Biotech). RNA blots were hybridized using mHR23A and .beta.-actin
.sup.32P-labeled cDNA probes.
Immunoblot analysis was performed on fibroblast extracts obtained
by sonification (5.times.10.sup.6 cells in 300 .mu.l
phosphate-buffered saline (PBS)) or extraction. In the latter case,
NP lysis buffer (25 mM Tris-HCl (pH 8.0), 1 mM EDTA, 10% glycerol,
0.01% Nonidet P-40, 1 mM dithiothreitol, 0.25 mM
phenylmethylsulfonyl fluoride, and protease inhibitor mix
(chymostatin, leupeptin, antipain, and pepstatin A)) was added to a
monolayer of MEFs. After 30 minutes on ice, the lysate was
collected with a cell scraper and clarified by 2 times
centrifugation at 4.degree. C. NP lysis buffer containing 0.3 M
NaCl was added to the cell pellet and homogenized by
sonification.
SDS polyacrylamide gel electrophoresis was performed by loading
25-50 .mu.g of total cellular protein per lane on 6-8% gels.
Proteins were blotted to nitrocellulose membranes (Schleicher &
Schuell) and probed with polyclonal antibodies recognizing human
HR23A or XPC, or with monoclonal antibodies recognizing the HA
epitope (HA.11, BAbCO) or p62 subunit of TFIIH (C39, kindly
provided by Dr. J. M. Egly). Proteins were visualized using
alkaline phosphatase-labeled goat anti-rabbit or
peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary
antibodies.
Immunofluorescence Labeling
Cells were grown on glass coverslips at 60-80% confluency. After
washing twice with PBS, cells were fixed with 2% paraformaldehyde
in PBS for 10 min at room temperature (RT) and permeabilized with
0.1% Triton X-100 in PBS for 2.times. 10 min at RT. After extensive
washing (three times of 5 min each) with PBS.sup.+ (PBS
supplemented with 0.15% glycine and 0.5% BSA), cells were incubated
with affinity-purified primary antibodies in PBS.sup.+ in a moist
chamber for 11/2 hr at RT. After washing five times in PBS.sup.+,
cells were incubated with the secondary antibodies for 11/2 hr in
PBS.sup.+ in a moist chamber at RT. Following 5 washes with
PBS.sup.+ and once with PBS, coverslips were preserved with
VECTASHIELD.RTM. Mounting Medium (Vector Laboratories) containing
4'-6-diamidino-2-phenylindole (DAPI, 1.5 .mu.g/.mu.l) to visualize
the nuclei.
Primary antibodies used: affinity-purified, rabbit polyclonal
anti-human XPC; rabbit polyclonal anti-human ERCC1; rabbit
polyclonal anti-XPA (a kind gift from Dr. K. Tanaka); mouse
monoclonal anti-p62 of TFIIH subunit (3C9, J. M. Egly, Illkirch);
and high affinity, rat monoclonal anti-HA (3F10, Boehringer).
Secondary antibodies were: goat anti-rat and goat anti-rabbit Alexa
594-conjugated, and goat anti-rat and goat anti-rabbit Alexa
488-conjugated antibodies (Molecular probes); and goat anti-mouse
Cy3-conjugated antibodies (Jackson ImmunoResearch
Laboratories).
Generation of XPC-GFP Fusion cDNA Construct and Cotransfection
Studies
Full-length human XPC cDNA (ScaI-Asp718I fragment) was cloned in
EcoRI-Asp718I digested eukaryotic expression vector pEGFP-N3
(Clontech) containing a 3' histidine-hemagglutinin tag (generated
by insertion of a double-stranded oligonucleotide in SspBI-NotI
digested pEGFP-N3; kindly provided by D. Hoogstraten). For
simplicity, the resulting tagged cDNA construct
hXPC-EGFP-His.sub.6HA-N.sub.3 is referred to as hXPC-GFP.
Full-length cDNAs of the hHR23B (in a pSLM vector, Pharmacia
Biotech) and hXPC-GFP were cotransfected into DKO MEFs using
puromycin as a selectable marker. The transfection was performed
using SUPERFECT.RTM. Transfection Reagent (Qiagen) and puromycin
was added 24 hr after transfection to a final concentration of 1
.mu.g/ml, and the cells were maintained under selection for 20-40
days. Stable puromycin-resistant clones were isolated and
integration of the cDNA construct was confirmed by DNA blotting
(data not shown).
Exposure of Cells to DNA Damaging Agents
Cells stably expressing hXPC-GFP/hHR23B were rinsed with PBS,
exposed to UV-C light (254 nm; Philips TUV lamp, dose as indicated
in the text) and subsequently cultured at 37.degree. C. for various
time periods (as indicated in the text). XPC was detected either by
immunoblot analysis or by visualization in living cells using
fluorescence microscopy. A similar approach was used to study the
effect of N-acetoxy-2-acetylaminofluorene (NA-AAF, final
concentration 50 or 100 .mu.M), mitomycin C (MMC, Sigma, final
concentration 1.2 or 2.4 .mu.g/ml), ionizing radiation
(.gamma.-rays from a .sup.137Cs source, single dose of 6 and 10
Gy), the proteasome inhibitor N-CBZ-LEU-LEU-LEU-AL (CBZ-LLL, Sigma,
final concentration 5 or 10 .mu.M), the transcription inhibitor
5,6-dichloro-1.beta.-D-ribofuranosyl-benzimidazole (DRB, Sigma,
final concentration of 100 .mu.M, 2-3 hrs), the translation
inhibitor cyclohexamide (CHX, Boehringer, final concentration 30,
50, and 100 .mu.g/ml, 1-3 hours), heat shock (39.5 and 41.degree.
C., for 2-12 hrs), and the nuclear export inhibitor leptomycin B
(LMB, Sigma, final concentration 10 ng/ml).
Local UV irradiation was obtained by covering cells grown on glass
coverslips with an isopore polycarbonate filter with pores of 5.0
.mu.m diameter (Millipore, TMTP) during UV irradiation (4.times.16
J/m.sup.2 UV-C). Immediately after exposure, the filter was removed
and medium was added back to the cells and culturing was continued.
After various time periods (as indicated in the text), cells were
processed for immunolabeling.
To identify cells in mixtures of control and mutant fibroblasts,
cells were labeled with latex beads (diameter 0.79 .mu.m,
POLYBEAD.RTM. Carboxylate Microspheres, Polysciences) added to
fibroblasts cultures 2 days prior to mixing of the cells. Cells
were thoroughly washed in PBS (3.times.) before trypsinization to
remove the non-incorporated beads and seeded in a 1:1 ratio on
coverslips and cultured for 2 days.
Heterokaryon Nuclear-Cytoplasmic Shuttling Assay
The shuttling assay using heterokaryons was performed as described
(Borer et al., 1989). One day before cell fusion, DKO cells stably
expressing hXCPC-GFP/hHR23B and HeLa cells were seeded in a 1:1
ratio on coverslips. Six hours prior to fusion, cells were
irradiated with 10 J/m.sup.2 UV-C or treated with 10 .mu.M CBZ-LLL.
Cell-fusion was induced (after washing with PBS) by treatment with
50% polyethylene glycol 6,000 in HANKS.TM. (Gibco) for 2 min
followed by (3.times.) washing with PBS. Finally, cells were
cultured in fresh medium either supplemented with or without
leptomycin B (LMB, final concentration 10 ng/ml). Three to five
hours after fusion, cells were fixed with 2% paraformaldehyde and
immunostained with rat monoclonal anti-HA (to monitor the
XPC-GFP-His.sub.6HA protein) and rabbit polyclonal anti-human ERCC1
(to distinguish human nuclei from mouse nuclei, since it
specifically recognizes human ERCC1) and subsequently with
appropriate secondary antibodies (see above).
Light Microscopy and Image Analysis
Immunofluorescent microscopy images were obtained with either a
Leitz Aristoplan microscope equipped with epifluorescene optics and
a PlanApo 63.times./1.40 oil immersion lens or a Leica DMRBE
microscope equipped with epifluorescene optics and a PL Fluotar
100.times./1.30 oil immersion lens. For the detection of GFP-tagged
proteins in the living cell, an Olympus IX70 microscope equipped
with epifluorescence optics and Olympus PlanApo 60.times./1.40 oil
immersion lens was used. GFP images were obtained after excitation
with 455-490 and long pass emission filter (>510 nm). Cy-3
images were obtained after excitation with 515-560 and long pass
emission filter (580 nm).
Results
Generation of mHR23A-Deficient Mice and Cells
To generate a mouse model for mHR23A, a targeting construct was
used in which exons III to VI and part of exons II and VII
(encoding residues 55 to 288 of the mHR23A protein) were replaced
by the neomycin resistance marker. Gene targeting creates an mHR23A
allele encoding a severely truncated protein in which >85% of
the coding sequence is deleted (even truncating the UbL domain) and
thus can be considered a null-allele (FIG. 1A). Two correctly
targeted clones (obtained at a frequency of 16%, FIG. 1B) were used
for blastocyst injections. Heterozygous offspring from matings
between germ line chimeric males and C57BL/6 female mice was
intercrossed to generate homozygous mutant mHR23A animals (FIG.
1C), as well as day 13.5 embryos (E13.5) for isolation of mouse
embryonic fibroblasts (MEFs). Neither the mHR23A mRNA nor the 50
kDa mHR23A protein could be detected in mHR23A.sup.-/- MEFs (FIGS.
1D and 1E). The two independent mouse lines were biochemically and
phenotypically indistinguishable for all parameters tested.
mHR23A.sup.-/- Animals and MEFs are NER Proficient
We assessed key repair parameters in mHR23A.sup.-/- MEFs. As shown
in FIGS. 2A-2C, UV survival, UV-induced unscheduled DNA synthesis
(UDS), and RNA synthesis recovery after UV exposure were all in the
wild-type range, indicating that global as well as
transcription-coupled NER are unaffected, mimicking the situation
in an mHR23B mutant (Ng et al., 2002). These data show that mHR23A
and mHR23B are functionally redundant for NER in vivo.
In striking contrast to mHR23B.sup.-/- animals, mHR23A.sup.-/- were
born with Mendelian frequency and appeared indistinguishable from
wild-type and heterozygous littermates for all parameters tested
(including morphology, main pathology, and growth rate up to 16
months). mHR23A.sup.-/- male and female mice were fertile, and
their mating activity and litter size were normal. Apparently,
mHR23A is not essential for mouse development and mHR23B can
compensate for any additional functions of mHR23A.
Total mHR23 Deficiency is Incompatible with Animal Life
In order to investigate the effect of a total mHR23 deficiency,
mHR23A.sup.-/-/B.sup.-/- animals (hereafter referred to as "DKO"
for double knockout) were tried to be generated and to obtain
corresponding MEFs by double heterozygous matings. Remarkably, out
of 427 newborns analyzed, no DKOs were found (Table 1). This shows
that inactivation of mHR23A aggravates the severe developmental
defects caused by an mHR23B deficiency (Ng et al., 2002) to a level
incompatible with life. Whereas phenotypically normal
mHR23A.sup.-/-/B.sup.+/- mutant mice at Mendelian ratios (71/427
found and 83/427 expected) were obtained, surprisingly
mHR23A.sup.+/-/B.sup.-/- animals were not born (0/427). However,
isolate E13.5 mHR23A.sup.+/-/B.sup.-/- mutant MEFs were isolated
although they showed poor growth. Apparently, loss of even one
allele of mHR23A in a complete mHR23B null-background causes
lethality in embryogenesis.
To investigate embryonic lethality caused by a complete mHR23B
deficiency, embryos at various stages of development were isolated.
No DKO embryos were present at days 13.5 and 10.5, but
growth-retarded mHR23-deficient embryos were observed at day 8.5.
Importantly, three DKO MEF lines were isolated from E8.5 embryos
(3/43, see Table 1). Compared to wild-type and double heterozygous
mutant MEFs, these cells displayed reduced rates of proliferation,
which resulted in the loss of two lines. Nevertheless, one DKO cell
line was established after 30 weeks culturing, which permitted
functional characterization of a total mHR23B deficiency.
Total mHR23-Deficient Cells Show an XPC-Like Repair Phenotype
Cell survival experiments revealed that DKO MEFs are remarkably
similar to the unique NER phenotype of XPC.sup.-/- cells in terms
of UV survival (FIG. 2D), deficiency of UV-induced UDS and
proficiency of RNA synthesis recovery after UV exposure (FIGS. 2E
and F). In contrast, MEFs retaining only one mHR23A or mHR23B
allele were NER competent (FIG. 2D). Apparently, one out of four
mHR23 copies is sufficient for normal NER activity.
We have examined the status of the XPC protein in the DKO MEFs.
Interestingly, steady-state levels of XPC appeared strongly reduced
in DKO MEFs compared to wild-type and mHR23A.sup.-/- cells (FIG.
1E), as shown by comparative immunofluorescence (FIG. 3A) and
immunoblot analysis of cell extracts (FIG. 3B). Thus, in the
absence of both mouse RAD23 proteins, XPC is unstable.
hHR23B and hXPC-GFP Rescue the UV Sensitivity of DKO Cells
To provide direct evidence that the XPC-like phenotype of DKO cells
is specifically caused by the mHR23 defect, (human) hHR23B cDNA
into DKO MEFs were stably transfected. The UV sensitivity of DKO
cells hHR23B was only partly rescued, perhaps due to human-mouse
differences (FIG. 4A). Importantly, expression of hHR23B induced an
increase in the total amount of endogenous (mouse) mXPC, as shown
by both immunoblot (FIG. 4C, lane 4) and immunofluorescence
analysis (FIG. 4D).
Subsequently, double mutant MEFs that stably express (human) hXPC
were generated, tagged with GFP (and additional His6 (SEQ ID NO:21)
and HA tags)) (FIG. 4B), to allow direct observation in living
cells. Functionality of the hXPC-GFP was demonstrated after
microinjection and transfection of the cDNA construct in
XPC-deficient cells (data not shown). Although hXPC-GFP was
undetectable by fluorescence microscopy (FIG. 4E), stable
transformants (verified for the presence of hXPC-GFP cDNA by DNA
blotting) had largely regained wild-type UV resistance (FIG. 4A),
indicating that the repair defect was rescued. Introduction of
hXPC-GFP appeared to restore endogenous mXPC levels as shown by
immunoblot (FIG. 4C, lane 5) and immunofluorescence analysis (not
shown). Apparently, hXPC-GFP has a trans-effect on mXPC
stability.
To investigate the stabilizing effect of mHR23B on XPC, hHR23B with
hXPC-GFP cDNA was cotransfected into DKO cells. Stably transfected
clones exhibited wild-type UV resistance (FIG. 4A) and normalized
levels of endogenous mXPC (FIG. 4C, lane 6, and not shown). In
contrast to MEFs expressing only hXPC-GFP, a small fraction
(<10%) of the double cotransfected cells displayed green
fluorescent nuclei (FIG. 4F). This is due to a level of hXPC-GFP
expression below the detection limit since immunofluorescence using
anti-HA monoclonals revealed that the majority of the cells
expressed the tagged transgene (data not shown). These data show
that the cotransfected hHR23B acts as a stabilizing factor for both
hXPC-GFP and endogenous mXPC.
DNA Damage Causes Accumulation of hXPC-GFP
The hXPC-GFP/hHR23B DKO cell line provided a convenient tool to
monitor the effect of DNA damage on the XPC steady-state level and
mobility in living cells. Interestingly, UV irradiation (5 and 10
J/m.sup.2) strongly increased the percentage of green cells and the
intensity of the GFP signal. Kinetic analysis upon UV exposure
revealed a time-dependent reversible accumulation of XPC-GFP in the
majority of the cells (FIG. 5A); this was further illustrated by
monitoring individual cells in time after UV irradiation (FIG. 5C).
In addition, these findings were corroborated by immunoblotting of
whole cell extracts using antibodies against the HA epitope
attached to the GFP tag (FIG. 5B, lane 2) and anti-HA
immunocytochemistry (not shown). Since this phenomenon was specific
for DKO cells transfected with hXPC-GFP/hHR23B, these results show
that XPC levels are responsive to UV in an HR23-dependent
fashion.
To investigate whether XPC accumulation is specific for NER-type
DNA damage or just stress-related, cells were exposed to different
kinds of genotoxic agents. N-acetoxy-2-acetylaminofluorene (NA-AAF,
50 and 100 .mu.M), which induces bulky adducts processed by NER,
elicited a very potent UV-like response in all cells within 6 to 8
hrs (FIG. 5D). In contrast, .gamma.-rays (6 and 10 Gy) and
mitomycin C (MMC, 1.2 and 2.4 .mu.g/ml), inducing mainly strand
breaks and interstrand cross-links respectively (which are dealt
with by other repair pathways), failed to provoke detectable XPC
accumulation. Also heat shock (41.degree. C., analyzed for up to 12
hrs) failed to boost fluorescence. The possibility that UV and
NA-AAF evoke a general accumulation of protein was ruled out since
cells expressing GFP alone do not exhibit a significant increase in
fluorescence after genotoxic insults. This shows that lesions
specifically recognized by the NER pathway enhance the level of
HR23-dependent hXPC-GFP.
One of the direct consequences of UV- and NA-AAF-induced DNA damage
is a temporary block of transcription. To investigate whether
hXPC-GFP accumulation requires transcription or is induced by a DNA
damage independent blockage of transcription, mRNA synthesis in DKO
cells expressing hXPC-GFP/hHR23B was reversibly arrested by
incubation with 5,6-dichloro-1.beta.-D-ribofuranosyl-benzimidazole
(DRB, 100 .mu.M). No induction of XPC-GFP fluorescence was
observed: instead, preincubation with DRB (2-3 hrs) prior to UV
treatment prevented UV-induced XPC-GFP accumulation (data not
shown). Consistent with this result, no enhanced XPC fluorescence
was found in cells treated with the translational inhibitor
cyclohexamide (30 and 50 .mu.g/ml), demonstrating the requirement
for de novo RNA and protein synthesis. In non-challenged
conditions, the steady-state level of XPC remains low.
hXPC-GFP is Degraded Via Ubiquitin/Proteasome-Dependent
Proteolysis
To further examine the HR23-dependent XPC stabilization, DKO cells
expressing hXPC-GFP/hHR23B were incubated with the proteasomal
proteolysis inhibitor N-CBZ-LEU-LEU-LEU-AL (CBZ-LLL, 5 and 10
.mu.M) (Wiertz et al., 1996). Similar to UV irradiation and NA-AAF,
all cells displayed a striking XPC-GFP accumulation in time (FIG.
5E), which was reversible upon drug removal (not shown). Both
immunoblot analysis (FIG. 5B, lane 3) and immunocytochemistry using
anti-HA antibodies (not shown) confirmed the above observations.
These findings show that degradation of XPC-GFP occurs via
ubiquitin/proteasome-dependent proteolysis and that an agent
capable of at least in part inhibiting proteolysis can be detected
by determining whether XPC accumulates in a cell.
Application of Local UV Damage to hXPC-GFP Expressing Cells
To explore the mechanism by which hXPC-GFP is stabilized, a
recently developed method for induction of DNA damage in a
restricted part of the nucleus was employed. For this purpose, a
monolayer of DKO cells expressing hXPC-GFP/hHR23B was covered with
a UV light-shielding isopore polycarbonate filter (pore diameter
.about.5 .mu.m). Upon UV irradiation, only at the position of pores
is UV damage induced, as detected with antibodies that specifically
recognize CPD and 6-4PP lesions. These locations attract all NER
proteins tested thus far. Cells were fixed at different time points
after UV irradiation to allow simultaneous immunostaining with
antibodies against various proteins and GFP fluorescence microscopy
(FIGS. 6A, 6B). Non-irradiated nuclei and non-damaged regions
within partly irradiated nuclei serve as internal controls. Very
rapidly (<2 minutes) after UV exposure, GFP fluorescence and
anti-HA immunostaining revealed high local accrual of
hXPC-GFP(His.sub.6HA) in part of the nuclei, which colocalized with
XPA (FIG. 6A) and the p62 subunit of TFIIH (not shown). These
findings demonstrate that, in living cells, the GFP-tagged XPC
protein translocates very rapidly to sites containing UV
lesions.
If XPC stabilization only occurs when bound to the damage, an
increase in fluorescent signal selectively at the damaged sites
would be expected. On the other hand, with an (additional) overall
stabilization of hXPC, it is expected that, in time, a concomitant
increase of fluorescence over the entire nucleus (in addition to
the damaged area) will be observed in comparison to non-damaged
nuclei. The increase of hXPC-GFP (FIG. 6A) initially occurs only at
the locally damaged sites, but after two hours also in the
remainder of locally damaged nuclei, a clearly higher signal is
noted when compared to non-exposed nuclei in the vicinity (FIG.
6B). These findings demonstrate an overall intranuclear
stabilization of hXPC-GFP triggered by binding to lesions.
High Levels of XPC Mediate a Transient Enhancement of DNA
Repair
To investigate the biological consequence of DNA damage-induced
stabilization of XPC, the DNA repair capacity (UV-induced UDS) in
DKO cells expressing XPC-GFP/hHR23B prechallenged with UV light.
Five hours post UV irradiation (10 J/m.sup.2) were tested the mean
UDS level (as determined by 1 hr .sup.3H-thymidine pulse-labeling
immediately after a dose of 16 J/m.sup.2) was 1.5-fold increased
compared to cells assayed in parallel that were not pre-irradiated
(FIG. 7A). UV-induced XPC-GFP accumulation was confirmed
microscopically (data not shown) just prior to the UDS assay. The
increase in UDS is not derived from the additional effect of NER
still dealing with lesions remaining of the first UV dose, since in
a separate UDS experiment without the second UV irradiation, no
significant UDS was observed (not shown). These data demonstrate
that UV-induced accumulation of XPC-GFP causes a concomitant
increase in GG-NER. Enhanced repair by increased levels of XPC was
confirmed by microinjection of XPC-GFP cDNA into homopolykaryons of
wild-type human fibroblasts. Microinjected cells expressing XPC-GFP
(FIG. 7A, top right panel) exhibit a higher UDS compared to
neighboring, non-injected monokaryons (FIG. 7B). In contrast, when
a cocktail of XPC-GFP and hHR23B was injected, UDS in the majority
of the cells was significantly lower and injection of this cocktail
appeared highly toxic (data not shown). These data demonstrate that
large amounts of stabilized XPC (as a result of overexpressed
hHR23B) can reduce cell viability.
Sequestration of XPC in the Nucleus Caused a Reduced
Proteolysis
The findings above demonstrate that XPC levels are under tight
control in an HR23-dependent fashion. Close inspection of the XPC
sequence revealed several potential nuclear location (NLS) and
nuclear export (NES) signals (provisionally referred to as NES1,
NES2 and NES3, FIGS. 8A and B). It was therefore investigated
whether nuclear-cytoplasmic shuttling regulates XPC levels as
reported for several other short-lived proteins, such as p53 and
clock proteins (Sionov et al., 2001; Yagita et al., 2002) and
whether DNA damage influences this process.
Nuclear export occurs via the chromosome region maintenance 1
(CRM1)/Exportin1 system (Mattaj and Englmeier, 1998; Nigg, 1997).
To investigate whether XPC shuttles between nucleus and cytoplasm,
the effect of leptomycin B (LMB), an established specific inhibitor
of CRM1/Exportin1-mediated nuclear export (Formerod et al., 1997;
Fukuda et al., 1997), on the location of XPC-GFP was studied. Using
a heterokaryon nuclear-cytoplasmic shuttling assay (Borer et al.,
1989) with DKO cells stably expressing XPC-GFP/hHR23B fused to
human cells (HeLa), transport of the fluorescent protein (both in
the presence and absence of 10 ng/ml LMB) from mouse nuclei to
human nuclei can be monitored. Four hours after fusion, cells were
immunostained with anti-HA monoclonals to identify the fusion
protein (XPC-GFP-His.sub.6HA) and specific human ERCC1 antibodies
(that do not cross react to rodent ERCC1) to recognize HeLa nuclei.
As shown in FIG. 8C, 4 hr after fusion in the absence of LMB, the
nuclear pool of XPC-GFP induced by 10 J/m.sup.2 UV-C light (given 6
hr prior to cell fusion) in the DKO cells was exchanged with
non-irradiated human nuclei. Administration of LMB directly after
cell fusion prevented this exchange (FIG. 8D), showing that export
from the mouse nuclei was responsible for the accumulation of the
XPC-GFP in the untreated HeLa nuclei. A similar effect on the
XPC-GFP shuttling was observed when XPC-GFP accumulation was
provoked by 10 .mu.M CBZ-LLL treatment (not shown). Parallel to the
documented cases of p53 and clock proteins these findings
demonstrate that proteolysis of XPC involves a nuclear-cytoplasmic
shuttling mechanism.
Generation of Clones Provided with Murine HR23
The mHR23A/mHR23B double mutant cells are transfected with
complementing functional mHR23A or mHR23B cDNAs tagged with
versions of the GFP fluorescent marker to permit in vivo dynamic
studies. Additionally, the mouse HR23 cDNA's are provided with
other tags facilitating purification on the basis of affinity
chromatography. Because murine genes are used in this experiment,
full functional complementation is obtained, avoiding possible
interspecies differences and consequent incomplete or aberrant
correction of a primary defect. In some of the transfections, other
genes/cDNAs known to be binding partners of HR23A or HR23B, such as
XPC, MAG, p53, centrin tagged with compatible fluorescent markers
and affinity tags are included in the transfection. Clones selected
for stable expression of the co-transfected dominant selectable
marker are screened for functional complementation of an HR23
defect and for proper expression of the other co-transfected gene.
Clones are used for identifying the network regulated by the HR23
pathway and application for readout of genotoxicity and for general
cellular stress.
A unifying model for the findings on HR23, XPC and proteolysis is
depicted in FIG. 9. As the main initiator of GG-NER, XPC
constitutes an ideal focal point for the regulation of the entire
pathway, which involves HR23. Absence of HR23 proteins reveals that
XPC on its own is highly unstable due to proteolysis via the 26S
proteasome. Under normal conditions, HR23 complex formation with
XPC results in a significant reduction of XPC proteolysis and
consequently in increased steady-state levels of the protein
complex. This correlates with proficient GG-NER. Under conditions
of a high level of DNA damage, involvement in NER stimulates the
protective role of HR23. Particularly after prolonged higher damage
load, this leads to gradual up-regulation of XPC and consequently
the entire GG-NER pathway. This rheostat model for adapting XPC
levels to the amount of damage provides a novel type of regulation
of DNA repair capacity in eukaryotes.
TABLE-US-00001 TABLE 1 Genotype analysis of DKO
(mHR23A.sup.-/-/B.sup.-/-) embryos and offspring Expected* Stage
Analyzed (if Mendelian) Found E8.5 43 7 .sup. 3.sup.# E10.5 14 1.8
0 E13.5 77 9.1 0 Newborn 427 41.4 0 *Derived from different
mHR23A.sup.+/-/B.sup.+/- and mHR23A.sup.-/-/B.sup.+/- intercrosses
#One cell line established
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SEQUENCE LISTINGS
1
21121DNAArtificialprimer mHR23Ap1 1atgggacttg ggcataggtg a
21220DNAArtificialprimer mHR23Ap2 2tcttcagcca ggcctcttac
20325DNAartificialprimer anti-sense neo 3atctgcgtgt tcgaattcgc
caatg 25421DNAArtificialprimer mHR23Bp1 4gtaaaggcat tgaaagagaa g
21521DNAArtificialprimer mHR23Bp2 5ctacagtctt gtttctgaca g
21620DNAArtificialprimer anti-sense pgk3 6taggggagga gtagaaggtg
20712PRTArtificialConsensus sequence NES 7Leu Xaa Xaa Xaa Leu Xaa
Xaa Xaa Xaa Leu Xaa Leu1 5 10811PRTArtificialhuman XPC NES-like
domain 1 8Leu Leu Pro Val Lys Pro Val Glu Ile Glu Ile1 5
10911PRTartificialmouse XPC NES-like domain 1 9Asp Met Pro Val Lys
Ala Val Glu Ile Glu Ile1 5 101027PRTArtificialmouse and human XPC
NES-like domain 2 10Leu Val His Ile Phe Leu Leu Ile Leu Arg Ala Leu
Gln Leu Leu Thr1 5 10 15Arg Leu Val Leu Ser Leu Gln Pro Ile Pro Leu
20 251130PRTArtificialhuman XPC NES-like domain 3 11Val Tyr Leu Phe
Leu Pro Ser Met Met Pro Ile Gly Cys Val Gln Leu1 5 10 15Asn Leu Pro
Asn Leu His Arg Val Ala Arg Lys Leu Asp Ile 20 25
301230PRTArtificialmouse XPC NES-like domain 3 12Val Tyr Leu Phe
Leu Pro Ser Met Met Pro Val Gly Cys Val Gln Met1 5 10 15Thr Leu Pro
Asn Leu Asn Arg Val Ala Arg Lys Leu Gly Ile 20 25
301327PRTArtificialmouse mXPC NES-like domain 2 13Leu Val His Ile
Phe Leu Leu Ile Leu Arg Ala Leu Gln Leu Leu Thr1 5 10 15Arg Leu Val
Leu Ser Leu Gln Pro Ile Pro Leu 20 25149PRTArtificialmouse
mXPC/hXPC NES-like domain 2 conserved region 14Leu Leu Ile Leu Arg
Ala Leu Gln Leu1 5158PRTArtificialmouse mXPC/hXPC NES-like domain 2
conserved region 15Leu Ser Leu Gln Pro Ile Pro Leu1
5169PRTArtificialmouse mXPC/hXPC NES-like domain 2 conserved region
16Leu Val His Ile Phe Leu Leu Ile Leu1 51711PRTArtificialmouse
mXPC/hXPC NES-like domain 2 conserved region 17Leu Gln Leu Leu Thr
Arg Leu Val Leu Ser Leu1 5 10189PRTArtificialmouse mXPC/hXPC
NES-like domain 3 conserved region 18Met Met Pro Ile Gly Cys Val
Gln Leu1 51911PRTArtificialmouse mXPC/hXPC NES-like domain 3
conserved region 19Val Tyr Leu Phe Leu Pro Ser Met Met Pro Val1 5
102010PRTArtificialmouse mXPC/hXPC NES-like domain 3 conserved
region 20Leu Asn Arg Val Ala Arg Lys Leu Gly Ile1 5
10216PRTArtificialHis6 tag antibody 21His His His His His His1
5
* * * * *
References